Systemic Inflammation and the Increased Risk of Inflamm-Aging and Age-Associated Diseases in People Living With HIV on Long Term Suppressive Antiretroviral Therapy

Abstract

The ART program in low- and middle-income countries (LMIC) like India, follows a public health approach with a standardized regimen for all people living with HIV (PLHIV). Based on the evidence from high-income countries (HIC), the risk of an enhanced, and accentuated onset of premature-aging or age-related diseases has been observed in PLHIV. However, very limited data is available on residual inflammation and immune activation in the populations who are on first-generation anti-HIV drugs like zidovudine and lamivudine that have more toxic side effects. Therefore, the aim of the present study was to evaluate the levels of systemic inflammation and understand the risk of age-associated diseases in PLHIV on long-term suppressive ART using a large number of biomarkers of inflammation and immune activation. Blood samples were obtained from therapy naïve PLHIV (Pre-ART, n = 43), PLHIV on ART for >5 years (ART, n = 53), and HIV-negative healthy controls (HIVNC, n = 41). Samples were analyzed for 92 markers of inflammation, sCD14, sCD163, and telomere length. Several statistical tests were performed to compare the groups under study. Multivariate linear regression was used to investigate the associations. Despite a median duration of 8 years of successful ART, sCD14 (p < 0.001) and sCD163 (p = 0.04) levels continued to be significantly elevated in ART group as compared to HIVNC. Eleven inflammatory markers, including 4E-BP1, ADA, CCL23, CD5, CD8A, CST5, MMP1, NT3, SLAMF1, TRAIL, and TRANCE, were found to be significantly different (p < 0.05) between the groups. Many of these markers are associated with age-related co-morbidities including cardiovascular disease, neurocognitive decline and some of these markers are being reported for the first time in the context of HIV-induced inflammation. Linear regression analysis showed a significant negative association between HIV-1-positivity and telomere length (p < 0.0001). In ART-group CXCL1 (p = 0.048) and TGF-α (p = 0.026) showed a significant association with the increased telomere length and IL-10RA was significantly associated with decreased telomere length (p = 0.042). This observation warrants further mechanistic studies to generate evidence to highlight the need for enhanced treatment monitoring and special interventions in HIV-infected individuals.

Keywords: HIV; India; LMIC (lower middle income country); inflammation markers; long term antiretroviral therapy.

Figure 1

Flow diagram of study design and experimental plan. 424 HIV-1 positive individuals and 295 HIV-1 negative healthy controls were screened. Following defined inclusion and exclusion criteria, 43 healthy controls, and 53 HIV positive ART-experienced subjects and 41 ART-naïve HIV-1 positive subjects were recruited for the study. The methodology used for the study is also presented.

Figure 2

Plasma immune activation markers. Soluble CD14 (A) and CD163 (B) in plasma of the three groups of individuals were measured using ELISA.

Figure 3

Plasma inflammation markers. (A) The random forest (RF) analysis of soluble factors resulted in predictive accuracies of 86.6% for HIVNC, Pre-ART, and ART. The soluble factors importance plots display the top 30 metabolites, which most strongly contribute to the groups' separation for HIVNC, Pre-ART, and ART. (B) Principal component analysis using PCAtool indicating grouping in different disease categories. (C) Hierarchical clustering analysis of ANOVA of top 20 differentially expressed proteins with false discovery rate (FDR) <0.001.

Figure 4

Comparative analysis of the proteins that differed significantly between groups. (A) Proteins that have statistically significant difference (p < 0.05, Tukey HSD) (in dark blue shade) between at least one of the groups compared (ART vs. Pre-ART, Pre-ART vs. HIVNC and ART vs. HIVNC). (B) Venn diagram showing significantly different protein in the study group. The sum of the numbers in each large circle represents the total number of differentially expressed proteins in plasma in the different groups (HIVNC vs. ART, Pre-ART vs.ART, and Pre-ART vs. HIVNC). The overlapping part of the circles represents significantly different proteins in the indicated groups. (C) Comparative analysis of 11 soluble markers that are significantly different between ART and HIVNC. (D) Heatmap and clustering of significantly different proteins on their protein-protein pair relation by Spearman-correlation in ART patients. ^*p < 0.05, ^**p < 0.001.